Method of producing brucella abortus vaccine



United States Patent 3,184,384 METHQD CF PRODUCING BRUCELLA ABORTUSVACCINE Joseph W. Whalen, Zionsville, Ind., assignor to The Dow ChemicalCompany, Midland, Mich, a corporation of Delaware No Drawing. FiledSept. 7, 1961, Ser. No. 136,453 5 Claims. (Cl. 167-78) This invention isconcerned with a method for preparing Brucella abortus vaccine and withan improved stabilized vaccine composition.

For a number of years, strain 19 of the Brucella abortus organism hasbeen employed for the preparation of vaccines useful for theimmunization of cattle against attack by virulent Brucella infections.In order to obtain proper immunization, large numbers of the live strain19 organisms must be injected. In the preparation of such vaccines, thepresence of appreciable numbers of dead or dissociated Brucellaorganisms is highly undesirable. It is also necessary to maintainculture conditions so that both virulent and non-irnmunogenic variantsof the desired strain 19 organism are avoided. Dissociation has been aparticular problem in prior attempts to culture Brucella abortus inliquid media.

In the past, the only approved method for the production of strain 19Brucella vaccines has involved culturing the Brucella organisms in acontainer such as a Roux flask or Povitsky bottle on a conventionalsolid potatoglycerol agar medium. In such cultures it has beenessential, in order to obtain the required yield of undissociatedorganisms, to avoid contact of liquids, such as the condensate whichnormally forms in such bottles, with the growing bacterial colonies. Thepreparation of vaccine in such manner requires the pooling of thewashings from the agar medium in a large number of such bottles, withattendant danger of contamination inherent in the number of operationsrequired.

Recently, the culture of Brucella abortus in liquid medium for theproduction of vaccine has been reported by Sterne, J. Gen. Microbiol.18, 747-750 (1958) and by Van Drimmelen, Onderstepoort Journal ofVeterinary Research, vol. 27, No. 4, December 1958. However, it has notbeen found possible to produce desirable numbers of Brucella abortusorganisms in a short time by the methods disclosed by Sterne and VanDrirnmelen. Also, it has been difficult to produce high yields ofundissociated organisms by said methods.

It is an object of the present invention to provide a method forproducing a vaccine concentrate of Brucclla abortus strain 19 of highpotency. It is a further object to produce such vaccines having enhancedstability. Other objects will become apaprent from the followingspecification and claims.

In accordance with the present invention, it has been discovered that bythe use of a particular liquid nutrient medium and cultural procedure,as hereinafter described, the organisms of Brucella abortus strain 19can be rapidly reproduced in said medium to provide a vaccineconcentrate of high potency. It has further been discovered that by theincorporation in said liquid culture medium of a particular stabilizersystem, both liquid and desiccated vaccines of unexpected stability areobtained.

In carrying out the invention, the liquid culture medium is prepared bydissolving a particular mixture of amino acids and peptides in watertogether with glucose, yeast extract, sodium chloride, thiaminehydrochloride and an antifoam agent, preferably a polypropylene glycol.This medium is sterilized, preferably by filtration, and is theninoculated with seed obtained by washing of smooth colonies of Brucellaabortus strain 19, propagated in corn ventional fashion on solidpotato-glycerol agar medium.

3,184,334 Patented May 18, 1955 The incubation of the Brucella organismsis carried out in a suitable vessel, such as a glass, glass-lined orstainless steel fermentor tank, equipped with a high-speed agitator anda sparger for introducing sterile air. Incubation of the organisms iscarried out with vigorous agitation at a temperature of 36 to 38 C.,while introducing sterile air at the rate of at least 7 volumes of airper minute for each 10 volumes of liquid culture medium. Culture of theorganisms under the foregoing conditions is continued for a period ofabout 20 hours for a single stage or up to about 40 hours whenadditional liquid medium is supplied as hereinafter described. In anycase, the incubation is continued for a sufficient period to obtainoptimal growth of viable organisms, and the resulting vaccineconcentrate is thereafter harvested by withdrawing it from the fermentorunder pressure into a sterilized, stainless steel holding tank or intosterile glass bottles. The bulk vaccine concentrate may thereafter bemixed with buffered physiological saline solution or other suitablediluent to prepare an injectable fluid vaccine product, or theconcentrate may be combined with a stabilizer and desiccated byfreeze-drying in conventional fashion to prepare a desiccated productsuitable for subsequent reconstitution with physiological salinesolution to produce an injectable vaccine.

The particular mixture of amino acids and peptides, found to beessential as a constituent of the nutrient medium employed herein forobtaining high yields of Brucella organisms, is a pancreatic enzymehydrolysate of casein containing, in the form of mixed amino acids andpeptides, all of the amino acids originally present in casein. Withcasein as normally derived from natural sources, said mixture alsocontains an appreciable por tion of the vitamins required for propergrowth of the Brucella organisms. Commercially available pancreaticenzyme hydrolysate of casein, suitable for use in accordance with theinvention, is available under the name of N-Z-Amine, Type A, from theSheflield Chemical Company. A representative analysis of the latterproduct is as follows.

Percent Total nitrogen 12.8 Amino nitrogen 6.8 Percent Amino N to totalnitrogen 53.0 Moisture 3.1 Lactose, maximum 1.5 Ash 5.2 pH (2% solutionin water, 25 C.), 6.6. Amino acid content (moisture-free basis):

Lysine 7.6 Isoleucine 6.4 Leucine 10.5 Valine 7.0 Arginine 3.3 Threonine4.0

Methionine 2.5 Cystine 0.3 Phenylalanine 4.5 Histidine 2.4

Tryptophane 0.9 Glutamic acid 20.

Vitamin content: Micrograms per gram Riboflavin 4.25 Thiamine 0.11Niacin 4.50 Pantothenic acid 2.19

Biotin 0.51 Pyridoxin 1.29

The choice of antifoam agent also appears to be important for obtainingdesirably high rates of production and high yields of the Brucellaorganisms. Thus, for example, the use of conventional antifoamagentssuch as corn oil, a commercial silicone antifoam agent (Dow- CorningAnti-Foam A) or the polyoxyethylene derivative of ricinoleic acidemployed by Van Drimmelen, while ef-- Ingredient: Percent by weightPancreatic hydrolysate of casein 3 Glucose q '2 Thiamin hydrochloride0.000 Yeast extract 1 Sodium chloride 0.5

Polypropylene glycol (molecular weight:

Demineralized water to make 100.00.

If desired, the polypropylene gycol antifoam agent can be incorporatedinto the inoculum seed suspension in amount to provide the foregoingconcentration when said seed suspension is mixed with the liquidnutrient-from which the polyglycol hasbeen omitted. It will be-apparcutto those skilled in the art that minor variations in the above amountsand proportions may be made without departing from the essence of theinvention." Any substantial departure from such composition,however,results in poor yields of vaccine or undesirable production ofdead or disassociated organisms. 7

' The foregoing proportions of ingredients produce a nutrient mediumhaving a pH of about 6.3 to 6.4. During the incubation of the Brucellaorganisms in said.

medium, the pH normally rises to a value of 8.1 to 8.3.

In general, the culture medium is maintained in this desired rangewithout further additaments. If, however, the

pH of the medium rises above the foregoing limits before the desiredamount of reproductive growth of the organisms is attained, smallamountsof sterile aqueous 50 percent glucose solution can be added tothe medium to maintain the pH in the desired range. V V 7 It has furtherbeen found that aeration of the growth medium during the incubation ofthe Brucella organisms by passing air into the upper part of thefermentor and relying on the mixing action of the agitator forintroducing air. into the medium, does not provide for the rapid growthof the organisms as embodied in the present invention. Accordingly, ithas been found essential to introduce sterile air into the lower portionof the fermentor tank below the surface of the nutrient mixture by meansof a sparger, or other suitable distribution device,

at the rate of at least'about 7 parts by volume of air per minute foreach '10 parts by volumeof culture medium.

In practice, the agitation of the nutrient liquid and the rapid growthprocesses of the organisms during the incubation period produce heat 'sothat cooling of the culture is usually required during at least part ofthe active growth.

period. Further, it is generally desirable to carryout themultiplication of the organisms in two or more stages. Conveniently, theseed inoculum harvested from the potato-glycerol agar intophosphate-buffered physiological saline solution is assayed and testedfor purity before use.

The seed inoculum is then introduced into a volume of the sterile liquidnutrient medium, as set forth above,

in proportionsof about 1 volume of inoculum to at least. about 14volumes of said medium, and the resulting mix-' ture is incubated underthe above-described conditions for a preliminary period of about 20hours to produce a working seed'culture. In such operations, the'numwasprepared as follows:

her of bottles of inoculum harvested and pooled is adjusted to provide asufficient concentration of Brucella organisms in the seed inoculum tofurnish an initial concentration of at least about 6x10 organisms permilliliter in the liquid nutrient medium, after admixing the inoculumtherewith. On completion of' said preliminary culture period, and whilevigorous multiplication of the Brucella organisms is still proceedingin'the working seed culture, a volume of fresh, sterile liquid nutrientmedium in amount equal to or up to twicethe volume of the working seedculture is run into the fermentor, and the incubation is then continuedunder the same'conditions for a further period of from about 15 to 20hours to complete the production of the desired vaccine concentrate.

The following examples illustrate the invention but are not to beconstrued as limiting the same.

. I Example 1 Brucella abortus organisms of strain 19 were inoculatedonto potato-glycerol agar contained in Povitsky bottles and incubated inconventional fashion. After incubation for 48 hours at '36- 38 C., theorganisms were harvested from 15 to 20 such bottles by washing withphysiological saline solution containing a phosphate buffer system tomaintain a pH of 6.4 and the washings were pooled, assayed and testedfor purity. The volume of washings and number of bottles harvested wasadjusted to provide an inoculum seed pool containing at least about 7510 viable Brucella organisms. Meanwhile, a fluid medium PercentPancreatic hydrolysate of casein 3 Glucose 2 Thiamine hydrochloride0.0005 Yeast extract 1 Nacl H 0.5 Polyglycol P-QOOO 2 0.0116 Water a93.4

vested as set forth above was then added to the fluid medium withagitation to provide an initial charge containing from about 06 x10 to1.5 x10 organisms per milliliter, and the resulting mixture wasincubatedat temperatures of 36,38 C. with the agitator operating at 240 .to 250revolutions perminute about 20 hours. During the above incubationperiod, sterile air was sparged into the lower part of the medium at arate of about 7.1 liters per minute for each 10 liters of said medium,and the temperature was maintained in the above range by heating orcooling as required. On completion of the incubation of this workingseed batch, a

further 175 liters of the above-described fluid medium was sterilized byfiltration and. passed into the fermentor tank. Incubation under theforegoing conditions was then continued for about 18 more hours, afterwhich the resulting'bulkvaccine concentrate'was harvested by drawing 01finto a sterilized, stainless steel holding tank and preserved bychilling to a temperature of 5 C. Serial dilutions of an aliquot of thisconcentrate were made with sterile aqueous .1 percent 'peptone solutionand suitable dilutions plated in Petri dishes on sterile potato agarcontaining horse serum and incubated for hours at 369-38 C. Plate countsshowed that the bulk vaccine concentrate contained about 200x10 viableBrucella abortus organisms per milliliter. Other similar serialdilutions were plated on sterile potato agar without horse serum,incubated and observed for dissociation by the method described byMingle and Manthei in American Journal of Veterinary Research, 2 (3),181-190 (1941). The vaccine concentrate was found to be substantiallyfree of dissociated forms. Further batches of vaccine concentrateprepared by the above procedure gave consistent yields of 150x10 to 22010 Brucella abortus organisms per milliliter.

Both liquid and desiccated vaccines produced from vaccine concentratesprepared according to the method of Example 1, have shown excellentstability in storage and have been employed routinely for a number ofmonths for the immunization of calves with no reported breaks inimmunity thereafter.

When it is desired to preserve the vaccine product by desiccationthereof, it is preferred to add a stabilizer, such as sterile skim milkor reconstituted non-fat dry milk solids, to the vaccine concentrateprior to freeze-drying thereof. In accordance with a further embodimentof the invention, it has been found that unexpected increased stabilityis obtained by employing a stabilizer of the following composition.

Ingredient: Percent by weight Potassium citrate monohydrate 0.135 Sodiumcitrate 0.245 Dipotassium acid phosphate 0.061 Calcium chloride 0.133Magnesium chloride hexahydrate 0.06 Potassium carbonate sesquihydrate0.1 Lactose 17.25

Water to make 100.00.

The pH of the foregoing composition, hereinafter referred to ascitrate-lactose stabilizer, was adjusted to 7, with lactic acid and thesolution sterilized by filtration before use.

The citrate-lactose stabilizer has also been found to be remarkablyeffective in the stabilization of liquid vaccines. Said stabilizerfurther appears to be safe for injections into cattle in the amountsemployed in normal dosages of vaccine.

The following examples illustrate improvement in stability of liquidvaccine obtained with the citrate-lactose stabilizer described above,and also the increased stability of vaccines prepared in accordance withthe present invention as compared with vaccines prepared by the cultureof the organisms on potato-glycerol agar using nonfat dry milk solids asstabilizer.

Example 2 A production lot of vaccine concentrate, prepared by themethod of Example 1, was divided into two batches and each batch dilutedsufficiently to produce an injectable vaccine containing a suitableconcentration of Brucella organisms. One such batch was diluted withsterile, phosphate-buffered, physiological saline solution at pH 6.4 inconventional fashion. The other batch was diluted with sterilecitrate-lactose stabilizer as described above. Portions of each batchwere assayed by serial dilution and plating in the usual manner todetermine the concentration of variable Brucella organisms in each batchas diluted. The batches were then maintained in closed containers at atemperature of 5 C. for six months and again assayed as before. Theresults are summarized in the following table, wherein the assays are interms of billions (10 of organisms per milliliter.

Viable Br. abortus organisms/ml. (X Diluent employed At; preparationAfter 6 months Phosphate-buttered saline 12. 9 6. 2 Citrate-lactosestabilizer 14. 5 13. 9

It will be noted that the vaccine prepared in conventional fashion withphosphate-buffered saline shows a loss during the storage period of over50 percent in the number of viable organisms per milliliter, while thevaccine stabilized in accordance with the present invention decreasedonly about 4 percent in titer.

Example 3 Ingredient: Percent by weight Vaccine concentrate Salinesolution 16 Milk solution 1 44 1 A sterile solution containing 92.6grams of Starlac brand non-fat dry milk solids per liter.

Further batches of vaccine concentrate were prepared in liquid mediumexactly as in Example 1. Two batches of such concentrates were dilutedwith physiological saline solution and reconstituted non-fat dry milksolution exactly as with the concentrates harvested from solid mediumabove. Two further such batches of concentrates produced in liquid mediawere diluted with the citratelactose stabilizer described above.

All batches of stabilized vaccines prepared as above were divided intostandard dosag volumes and the latter bottled and freeze-dried incommercial equipment. After desiccation, representative samples of eachbatch of vaccine were reconstituted, serially diluted and plated bystandard procedure to determine the concentration of viable organisms ineach batch. The bottled, desiccated vaccines were then placed in anincubator maintained at 37 C. Such Vaccines are normally keptrefrigerated until reconstituted for use. Thus, particularly with aliving vaccine such as is here involved, storage at 37 C. constitutes aseverely accelerated test of stability. Representative samples from eachbatch of vaccine were removed from the incubator after storage intervalsof 5, 10 and 20 days and reconstituted and assayed as before. Theresults are summarized in the following table, wherein the figuresrepresent billions of visable Brucella organisms per milliliter ofreconstituted vaccine.

Viable Brucella abortus organisms (bil./ml.) at indicated time intervalNutrient medium Stabilizer after desiccation Initial 5 days 10 days 20days Solid, potato agar Non-fat milk 16.2 12.5 6.0 0.3 Do "(10 l4. 3 12.5 6. 0 0. 3 Liquid, as in Ex. 1 10. 9 14. 2 10. 5 0. 3 Do... 24.1 19. 78.3 7. 2 D0- 19. 9 16. 5 14. 8 9. 0 D0- 21. G 17. 8 15. 3 l2. 5

The Initial figures in the vpreceding tableare the values for thesamples reconstituted andassayed immedi-' ately after desiccation. Theresults show not only the increased stability of vaccines stabilizedwith the, citrate-- lactose stabilizer, but also the increased stabilityof the vaccine produced by the liquid medium process of the presentinvention when stabilized with non-fat milk in comparison with similarlystabilized vaccine producedlby the accepted prior art method ofculturing on solid potato agar.

The vigorous growth of the organisms and lack of dissociation thereof inthe liquid nutrient medium process of the invention makes said processadaptable as a con tinuous or semicontinuous process. In such a process,a portion of the liquid culture is Withdrawn from the fermentor prior tocompletion of the production of the vac cine concentrate and while theorganisms are reproducing vigorously at a logarithmically increasingrate of growth. Said portion is then employed as the inoculum seed in 'afurther fresh volume of the liquid nutrient medium and the furtherculture of the organisms is continued as before for as many cycles .asdesired.

I claim: I i a 1. The method for producing a vaccine concentrate ofBrucella abortus organisms which comprises preparing a sterile liquidnutrient medium consisting of an aqueous solution of yeast extract,sodium chloride, thiamine hydrochloride, a polypropylene glycol having amolecular weight of from about 1500 to 3000 as an anti-foaming agent 2percent by weight of glucose and about 3 percent by weight of apancreatic enzyme hydrolysate of casein,

seeding .said solution with a suspension of organisms of strain 19 ofBrucella abortus, agitating the resulting mixture and maintaining sameat temperatures of 36'38 C., while introducing sterile air below thesurface of said mixture at the rate of at least 7 volumes of air perminute for each 10 volumes of liquid culture medium, maintain ing theforegoing incubation conditions for a period of from about 20 to hoursand harvesting the resulting vaccine concentrate.

2. A method according to claim 1 wherein themedium has the followingcomposition:

Ingredient: Percent-by weight Pancreatic hydrolysate of casein ,3Glucose 2 Thiamine hydrochloride 0.0005

Yeast extract 1 claim 4.

Percent by weight Sodium chloride 0.5 Polypropylene glycol (molecularweight:

000) 0.0116 Demineralized water to make 100.00.

'3. 'A method according to claim 1 wherein the inoculum of Brucellaorganisms is-introduced into a volume of the nutrient medium in anyamount to provide from about 0.6x 10 to 15 x19 viable Brucella organismsper milliliter. in the resulting mixture, said mixture is incubated inaccordance with the method of claim 1 for a preliminary period of about20 hours, a further'volume, at least equal 'to the amount of theinitialnutrient medium employed, of fresh, sterile nutrient medium isintroduced and incubation continued in the same fashion for a furtherperiod of from about 15 to 20'hours to complete the production of thedesired vaccine concentrate.

. -4. A method according to claim 1 wherein the vaccine concentrateproduct is mixed with astabilizer consisting essentially of an aqueoussolution containing in parts by weight 135 parts potassium citratemonohydrate, 2 45 parts sodium citrate, 61 parts dipotassium acidphosphate, 133 parts calcium chloride, parts magnesium chloridehexahydrate, parts potassium carbonate sesquihydrate and 17,250 partslactose per.100,000 parts of Water.

IS. A stabilized vaccine prepared by the method of 6. A method accordingto claim 4 wherein the stabilized vaccine product is freeze-dried.

References Cited bythe Examiner I UNITED STATES PATENTS 2,682,492 6/54Huddleson 16778 OTH R REFERENCES LEWIS'GOTTS, Primary Examiner. M. o.WOLK, IRVING MARcUsExmnmers.

1. THE METHOD FOR PRODUCING A FACCINE CONCENTRATE OF BRUCELLA ABORTUS ORGANISMS WHICH COMPRISES PREPARING A STERILE LIQUID NUTRIENT MEDIUM CONSISTING OF AN AQUEOUS SOLUTION OF YEAST EXTRACT, SODIUM CHLORIDE, THIAMINE HYDROCHLORIDE, A POLYPROPYLENE GLYCOL HAVING A MOLECULAR WEIGHT OF FROM ABOUT 1500 TO 3000 AS AN ANTIFOAMING AGENT 2 PERCENT BY WEIGHT OF GLUCOSE AND ABOUT 3 PERCENT BY WEIGHT OF A PANCREATIC ENZYME HYDROLYSATE OF CASEIN, SEEDING SAID SOLUTION WITH A SUSPENSION OF ORGANISMS OF STRAIN 19 OF BRUCELLA ABORTUS, AGITATING THE RESULTING MIXTURE AND MAINTAINING SAME AT TEMPERATURES OF 36*-38* C., WHILE INTRODUCING STERILE AIR BELOW THE SURFACE OF SAID MIXTURE AT THE RATE OF AT LEAST 7 VOLUMES OF AIR PER MINUTE FOR EACH 10 VOLUMES OF LIQUID CULTURE MEDIUM, MAINTAINING THE FOREGOING INCUBATION CONDITIONS FOR A PERIOD OF FROM ABOUT 20 TO 40 HOURS AND HARVESTING THE RESULTING VACCINE CONCENTRATE. 